Bacterial pathogens in humans and rodents are being recognized as potentially having a direct role in carcinogenesis. A number of bacterial toxins are known to modulate intracellular signaling pathways directly that could promote tumor development, while their carcinogenic potential is not fully established. Bacterial virulence factors that interfere with cell signaling and result in disruption of normal cell division and apoptosis could promote anchorage-independent growth and facilitates cancer metastasis. The mechanism of anchorage-independent growth could include production of growth factors that act as an autocrine or paracrine molecules, and change in the activity of signaling pathways. It is known that Pasteurella multocida toxin (PMT) treated cells were able to proliferate and showed a striking increase in the formation of colonies in soft agar. The catalytic domain of rPMT has been shown to catalyze the deamidation of a conserved glutamine residue in the alpha-subunit of the heterotrimeric G proteins, and inhibits its GTPase activity. Thus, deamidation converts these G proteins to a constitutively active phenotype and stimulates several signaling cascades, including those linked to PLC-beta, MAPK, and Ca(II) mobilization. However, the mechanism behind cell proliferation and the anchorage-independent growth due to rPMT treatment still remains to be established. We have shown that rPMT induces protein and ATP synthesis, cell migration and proliferation in serum-starved Swiss 3T3 cells. Concomitantly rPMT induces a sustained phosphorylation of ribosomal S6 kinase and its substrate, ribosomal S6 protein. This phosphorylation is inhibited by two specific inhibitors of mammalian target of rapamycin (mTOR), rapamycin and Torin1. Using monoclonal antibody against Gq and Gq/11 double-deficient fibroblasts, we established for the first time that PMT activates mTORC1 through a Gq/11/PLCbeta/PKC pathway, and the rPMT-induced protein synthesis and cell migration are, in part, mediated by the mTORC1 pathway. Furthermore, rPMT-treated cells are capable of generating and secreting into the medium compound(s) which can activate autocrine and/or paracrine signaling. This diffusible factor(s) activates mTOR and MAPK pathways independent of Gq/11. We also found that rPMT treatment greatly up-regulates the connective tissue growth factor (CTGF), an ECM protein which is known to regulate a diverse array of cellular functions including cell proliferation, migration and differentiation, in both mRNA and protein levels. The mechanism of this up-regulation is mediated by a MAPK-dependent pathway and independent of TGFbeta, a well-established CTGF inducer, and of the mTOR signaling pathway. To elucidate the respective roles of Gq and G12 proteins and their deamidated active forms in PMT-mediated cell signaling, we established several cell lines harboring wild type Gq, G12 along with GFP as a control using targeted PhiC31 integrase technology. PhiC31 integrase is a site-specific recombinase from a bacteriophage that has become a useful tool in mammalian genomic editing. The enzyme normally performs precise, unidirectional recombination between two attachment (att) sites called attB and attP. We first preintegrated an attP docking site (landing pad) into Hek 293 and Swiss 3T3 cellular chromosomes. The inserted attP site is uniquely recognized by a secondary, proprietary integrase of bacteriophage origin (Pin integrase). This attP site on the landing pad serves as a target for integration of a donor plasmid carrying an attB site and our gene of interest. In contrast to the PhiC31 integrase, which can recognize endogenous sequences that resemble attP sites (pseudo attP), the Pin integrase does not recognize pseudo sites, resulting in singular, site specific integration at the landing pad site. Subsequent recombination after landing pad integration, thus leads to precise integration of the plasmid into the chromosome at the attP site. This specificity is useful when establishing multiple cell lines because all genes introduced into the same landing pad site, standardizing the chromosomal environment in order to minimize position effects associated with random integration. After establishment of cell lines containing the landing pad site recognized by the Pin integrase, constructs for both the wild-type and constitutively active forms of the G proteins of interest, as well as for a GFP control were used to generate the desired cell lines. Subsequent to recombination, several clones were isolated and propagated. PCR analysis on genomic DNA revealed integration of the transgenes in the genome of Hek 293 and 3T3 cells. RT-PCR analysis and western blotting using appropriate antibodies indicated that both recombinant wild type and deamidated form of Gq, G12 and the GFP control were expressed in both Hek293 and Swiss 3T3 cells. Furthermore, to study the role Gq deamidation on Gq cellular localization, we established multiple cell lines expressing a Gq-GFP fusion construct in either the wild-type or deamidated form, using the same technology. Based on GFP visualization, both wild type and deamidated Gq are localized both in the cytoplasm and at the cell membrane, whereas a GFP-control was localized only to the cytoplasm. Unfortunately, all cell lines established, including the ones that expressing control GFP, in both Hek293 and 3T3 cells, showed a drastic decrease in transgene expression, ultimately leading to complete transgene inhibition after the cell lines were passaged several time. The inhibition of transgene expression was observed in both protein and the mRNA level. We speculate that the inhibition of the expression of transgene in our cell is probably due to the promotor silencing. It is worth mentioning that all studies related to the establishment of cell lines that overexpress heterotrimeric G proteins including Gq and G12 and their mutant forms do not mention the inhibition of the transgene expression as a function of the passage number of the cells. To overcome this problem, we established new cell lines for all genes of interest using the inducible Tet system in both Hek293 and 3T3 cells. This technology uses a different promoter sequence, thus circumventing potential promoter silencing. To this end, we established new cell lines for all genes of interest using Tet-On 3G inducible mammalian expression system that is controlled by adding doxycycline to the culture media. This technology uses a different promoter sequence, thus circumventing potential promoter silencing. It consists of 7 repeats of a 19 bp tet operator sequence located upstream of a CMV promoter. Western Blot analysis showed expression of our construct only upon addition of doxycycline. Additional Western Blot analysis shows an increase of total Gq upon induction of cell lines containing our wild-type construct. Control GFP and Luciferase cell lines also show no background expression when no doxycycline was added. Each cell line was then analyzed for expression of CTGF and ribosomal S6 phosphorylation, both hallmarks of PMT treatment. Unlike PMT treatment, expression of deamidated Gq does not result in CTGF production or mTOR activation (monitored by S6 phosphorylation). It was additionally shown that PMT treatment results in an increase in MAPK (Erk1/2) phosphorylation, but no increase is seen for wild type or deamidated Gq expression. It is clear from these experiments that overexpression of deamidated Gq did not recapitulate the biological effect of PMT in term of CTGF expression, mTOR and MAPK activation. However, the serum starved cell line that expressed deamidated Gq exhibit a similar shape relative to that exhibited with PMT treated serum-starved cell. PMT treated cells and Gq expressing cell are flat and spread out while control cells are round when they are serum-starved.